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Originally published online as doi:10.1189/jlb.0806528 on August 27, 2007

Published online before print August 27, 2007
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(Journal of Leukocyte Biology. 2007;82:1575-1584.)
© 2007 by Society for Leukocyte Biology

Neutrophil transepithelial migration in response to the chemoattractant fMLP but not C5a is phospholipase D-dependent and related to the use of CD11b/CD18

Svetlana O. Carrigan*, Desmond B. S. Pink{dagger} and Andrew W. Stadnyk*,{dagger},1

* Departments of Microbiology and Immunology and
{dagger} Pediatrics, and the Dalhousie Inflammation Group, Dalhousie University, Halifax, Nova Scotia, Canada

1Correspondence: Mucosal Immunology Research, IWK Health Centre, 8 West, 5850/5980 University Ave., Halifax, Nova Scotia B3K 6R8, Canada. E-mail: astadnyk{at}dal.ca


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ABSTRACT
 
In Crohn’s disease and ulcerative colitis patients, the numbers of neutrophils recovered from stool directly correlates with the severity of disease, implying that neutrophils in the lumen contribute to the tissue destruction; therefore, it is important to understand the mechanisms behind transintestinal epithelial migration. Neutrophil transintestinal epithelial migration to fMLP is appreciated to be CD11b/CD18 integrin (Mac-1)-dependent, while we recently reported that migration to C5a is Mac-1-independent. Here, we investigated whether phospholipase D (PLD), a signaling molecule linked to chemoattractant activation of neutrophils, is necessary for both Mac-1-dependent and Mac-1-independent migration. Both fMLP and C5a increased neutrophil expression of the Mac-1 activation epitope, indicating PLD was activated. This up-regulation was dose-dependently prevented by incubation of neutrophils in 1-butanol, an inhibitor of PLD activity. Despite this effect on Mac-1, 1-butanol did not prevent neutrophil migration across acellular filters. Incubation in 1-butanol did inhibit fMLP but not C5a-mediated migration across intestinal epithelial cell monolayers, showing that transepithelial migration to fMLP but not C5a is dependent on PLD. The addition of phosphatidic acid, a reaction product of PLD, partially restored fMLP-mediated transepithelial migration in the presence of 1-butanol but not the migration of Mac-1-deficient neutrophil-differentiated HL-60 cells. Thus PLD control over expression of the Mac-1 activation epitope is critical for neutrophil migration to fMLP but not C5a. Moreover, as PLD controls other neutrophil functions, such as the oxidative response, degranulation, and protease release, we could exclude these functions as being important in neutrophil transepithelial migration to C5a.

Key Words: leukocyte • mucosa • chemotaxis • signal transduction • lipid mediators


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INTRODUCTION
 
Crypt abscesses are a prominent histological feature of the active inflammatory bowel diseases, Crohn’s disease, and ulcerative colitis. In fact, the level of neutrophil products recovered in patient stool is directly related to disease severity, and granulocyte infiltration of the mucosa is a diagnostic criterion of disease relapse [1 , 2 ]. Drawing on these observations, an emerging opinion is that neutrophil accumulation in the crypt lumen, and not necessarily the mucosa, is important to perpetuating chronic inflammation by damaging the epithelium. This hypothesis is supported by experimental evidence that neutrophil accumulation in the mucosa in the absence of transepithelial migration does not necessarily cause damage [3 ] and by electron microscopic observation that migrating neutrophils have not degranulated [4 ]. We have consequently taken the approach that it is important to prevent neutrophil migration into the intestinal lumen to control epithelial damage and inflammation.

Neutrophil transintestinal epithelial migration to the bacterial product, fMLP, modeled in an in vitro system, is critically dependent on the β2 integrin, CD11b/CD18 (Mac-1) [5 ]. Yet a number of lines of evidence have suggested that other neutrophil chemoattractants might be present in the inflamed intestinal lumen and therefore likely contribute to recruiting neutrophils into and across the intestinal epithelium [6 , 7 ]. In the course of studying migration to C5a, IL-8 and LTB4 and using anti-CD11a, CD11b, and CD18 monoclonal antibodies, we showed that Mac1-independent (indeed, CD18-independent) mechanisms of neutrophil transintestinal epithelial migration exist [8 ]. Additionally, we showed that HL-60 cells differentiated with dbcAMP into neutrophil-like cells but which lacked Mac-1 surface expression, migrate across epithelial monolayers to C5a, further confirming that Mac-1-dependent interactions are not necessary for transepithelial migration in response to C5a [9 ]. As for in vivo evidence, CD11b-deficient mouse PMN infiltrate the inflamed mucosa in the dextran sodium sulfate model of colitis, and these animals actually experienced worse disease than wild-type [10 ]. Now we are interested in distinguishing the signaling events behind neutrophil Mac-1-dependent (in response to fMLP) vs. Mac-1-independent (in response to C5a) transepithelial migration and choose here to examine phospholipase D (PLD) signaling, considering the multiple roles this enzyme plays in PMN activation.

C5a and fMLP signal through Gi-coupled receptors, activating phospholipases, including PLD [11 ]. PLD, in turn, is important in multiple neutrophil functions including the respiratory burst [12 ], degranulation [13 ], elastase release [14 ], MAPK activation [15 ], phagocytosis [16 ], adhesion [17 , 18 ], apoptosis [19 ], and β2 integrin expression [20 ] (reviewed in [21 ]). Some known anti-inflammatory compounds, for example, resveratrol and lipoxins have been shown to decrease PLD activation, and diabetic patient susceptibility to infections may be explained by high glucose concentrations impairing PLD activation [22 ]. Thus, PLD is important in chemoattractant activation of neutrophils.

The role of PLD in leukocyte chemotaxis and migration is emerging with studies implicating PLD in the expression of activated Mac-1 by C5a in eosinophils [23 , 24 ] and by fMLP in neutrophils [18 ]. This is compatible with the role PLD plays in neutrophil and macrophage adhesion and chemotaxis, including under shear stress [18 , 25 , 26 ]. Although human neutrophils express multiple isotypes of PLD1 and PLD2, it appears that PLD1 is critical for chemotaxis, shown using RNA knockdown methods on granulocyte differentiated HL60 cells stimulated fMLP or IL-8 [26 ]. It is noteworthy that RNA knockdown approaches have not been used on primary neutrophils and instead alcohols remain the main means to inhibit PLD and to study the role of phosphatidic acid (PA) in primary neutrophil function. Alcohol substitutes for water in the transphosphatidylation reaction catalyzed by PLD and results in the generation of inactive phosphatidylalcohols instead of phosphatidic acid, therefore preventing PLD-dependent neutrophil activation. This reaction may explain why alcohol-intoxicated individuals had decreased neutrophil recruitment into skin abrasions, and further suggests that PLD plays a role in neutrophil migration [27 , 28 ].

Despite these advances in understanding the role of PLD in adhesion, it has not been studied in neutrophil transepithelial migration. We therefore aimed to determine whether PLD activation plays any role in distinguishing Mac-1-dependent vs. Mac-1-independent neutrophil migration across a model intestinal epithelium. We hypothesized that the PLD pathway is important when neutrophils use Mac-1 when activated by the chemoattractant fMLP but not C5a. In turn, results from these experiments will help predict the contribution of a number of other signaling mechanisms downstream of PLD in transepithelial migration.


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MATERIALS AND METHODS
 
Reagents and antibodies
rhC5a, fMLP, 1-butanol, tert-butanol, (±)-propranolol hydrochloride, and dbcAMP were purchased from Sigma Chemical Company (Oakville, ON, Canada). Phosphatidic acid (DiC8-PA) was obtained from Avanti Polar Lipids (Alabaster, AL, USA). Propranolols were diluted in DMSO and prepared fresh for each experiment. DiC8-PA was diluted in distilled water as a stock concentration of 50 mM. Mouse monoclonal anti Mac-1 activation epitope (clone CBRM1/5) [29 ] was purchased from EBioscience (San Diego, CA, USA). Monoclonal anti-PLD1 (IgG2a, clone F-12) was purchased from Santa Cruz (Santa Cruz, CA, USA).

T84 and HL-60 cell culture
T84 colon epithelial cells (American Type Culture Collection, Bethesda, MD) were cultured in 1:1 HAM F12/DMEM (Invitrogen, Grand Island, NY, USA) containing 5% newborn calf serum, 15 mM HEPES, 50 U/ml penicillin, and 50 µg/ml streptomycin (Life Technologies, Burlington, ON, Canada) and grown as inverted monolayers on TranswellTM filters (Costar, Corning, NY, USA), as described previously [9 ]. Briefly, 5 x 105 cells were seeded on the undersurface of 0.33 cm2 polyester Transwell filters (3-µm pore size) first coated with type I rat tail collagen (ICN Biomedicals, Aurora, OH, USA). Cells were allowed to adhere then placed upright into 24-well plates and grown for 7 or 8 days in complete HAM F12/DMEM. Monolayer permeability was measured one day before the migration experiment using 125I-conjugated to human serum albumin (HSA) applied to the top chamber and measuring radioactivity in the bottom chamber, and diffusion was routinely less than 1%. When filters were used without T84 monolayers (acellular filters), the filter was soaked in T84 media for 2 h before the addition of neutrophils.

The HL-60 promyelocytic cell line (American Type Culture Collection) was cultured in Iscove’s modified Dulbecco’s medium (Invitrogen), then to differentiate into the granulocyte lineage, cells at a concentration of 5 x 105 cells/ml were incubated with 0.5 mM dibutyryl cyclic AMP (dbcAMP) for 2 days, as previously reported [9 ].

Neutrophil isolation and labeling
Adult volunteers were recruited and bled for peripheral blood in compliance with the terms of the Research Ethics Board of the Health Centre. Briefly, and as described before [8 , 9 ], leukocyte-rich plasma was collected following dextran sedimentation of the blood, and the cells were labeled with sodium chromate (~375 mCi/ml, Na251CrO4; Amersham, Oakville, ON). The leukocyte fraction was then layered on a 58%/72% discontinuous Percoll (Pharmacia, Uppsala, Sweden) gradient and centrifuged; neutrophils were collected from the 58%/72% Percoll interface. The enriched neutrophils were washed 3 times in Ca2+ and Mg2+-free Tyrode’s solution and resuspended in DMEM supplemented with 5 mg/ml pyrogen-free HSA, 15 mM HEPES at a concentration of 106 cells/ml. Neutrophils were >95% pure by crystal violet dye staining and >98% viable by trypan blue dye exclusion.

dbcAMP differentiated HL-60 cells were resuspended in Ca2+ and Mg2+-free Tyrode’s, and incubated with Na251CrO4 for 30 min at 37°C. The cells were then washed 3 times in Ca2+ and Mg2+-free Tyrode’s buffer and resuspended in DMEM with 5 mg/ml HSA and 15 mM HEPES at a concentration of 106 cells/ml. Cells were ≥90% viable by trypan blue exclusion prior to the migration assay.

Migration assay
The migration assay was performed as described earlier [9 ]. Neutrophils or differentiated HL-60 cells were incubated with 1-butanol, tert-butanol, DiC8-PA, or (±)-propranolol hydrochloride as indicated in the figure legends prior to applying 105 cells in 100 µl to the upper chamber of the Transwell. The concentration of propranolol used, 300 µM, is the highest reported to have a specific effect on human neutrophils. Filters were placed into the wells of the 24-well plate containing chemoattractants in 600 µl of fresh DMEM/HEPES/HSA. Cells were collected after 70 min (across acellular filters) or 2 h (across T84 monolayers). We titrated the concentration of chemoattractants for acellular filters and epithelial monolayers to achieve the best rate of migration and observed plateaus within the first hour of migration across acellular filters and after 1.5 h across monolayers (data not shown). The optimal C5a concentrations for migration were 10–9 M and 10–8 M with acellular filters and T84 monolayers, respectively, for both neutrophils and HL-60 cells. For fMLP, 10–8 M and 10–7 M were optimal with acellular filters and T84 monolayers, respectively, for neutrophils, and 10–9 M and 10–8 M, respectively for HL-60 cells. Cells that migrated into the lower chamber were lysed with 1% Triton X-100 while monolayer-associated (adherent) cells were lysed with 0.2 M NaOH. Fractions were collected separately, and radioactivity (cpm) was determined using a Wizard 1480 automatic {gamma} counter (Wallac, Turku, Finland). The percent of applied cells or adherent cells was calculated as cpm of the migrated (or monolayer-associated) fraction/cpm of the cells added to the upper chamber at the beginning of the migration assay x100%. Triplicate wells for each treatment were performed, and data are reported as the means ± standard deviation (SD) when showing a representative experiment or mean of the mean % applied cells from several experiments ± SEM, as specified in the figure legend.

Immunoprecipitation
PMNs (1.2 x 107 cells/ml) were incubated with PBS containing 1.0 mM diisopropylfluorophosphate (DFP) for 20 min (all steps performed at 4°C) and then lysed in cold lysis buffer (50 mM HEPES, pH 7.5, 100 mM NaCl, 1 mM EDTA, 1 mM EGTA, 20 mM NaF, 1 mM Na3VO4, 1% Triton X-100, 1% deoxycholate, 0.3 mM AEBSF, 0.2 µM aprotinin, 5 µM leupeptin, 10 µM bestatin, 4 µM pepstatin-A, 4 µM E-64, phosphatase inhibitor cocktail 2 (20 µl/ml lysis buffer; Sigma). Cells were sheared using seven passes through a 20-gauge needle, rotated for 30 min, and finally centrifuged (5 min at 14,000 g) to remove insoluble material. Next, supernatants were precleared for 30 min using 1.0 µg normal mouse IgG and 20 µl Protein-A Sepharose, centrifuged (1000 g for 5 min) and 1 ml of the resulting supernatant combined with 2.0 µg of anti-PC-PLD1. Samples were rocked gently for 1.5 h, after which 20 µl of Protein-A Sepharose was added, and the mixture was rotated at 4°C overnight. Immunoprecipitates were centrifuged (1000 g for 5 min), washed 4 times with ice-cold PBS, and then resuspended in electrophoresis buffer (62.5 mM Tris, pH 6.8, 2% SDS, 10% glycerol, 0.1M β-mercaptoethanol). Immunoprecipitates were boiled for 5 min, subjected to 7.5% SDS-PAGE, and finally transferred to a Protran BA83 nitrocellulose membrane. Membranes were processed as described elsewhere [30 ] and probed using anti-PC-PLD1 and HRP-conjugated goat anti-mouse secondary antibody (1:2000, Santa Cruz).

Flow cytometry
Purified neutrophils at 106 cells/ml in DMEM/HSA/HEPES were treated with tert-butanol or 1-butanol or left untreated, plus CBRM1/5 or isotype control monoclonal antibody followed by an incubation with chemoattractant at concentrations 10x less than the optimal concentration for migration or DMEM for 30 min at 37°C. At the end of the incubation, the cells were rapidly cooled, washed with ice-cold HBSS containing 0.5% BSA and 0.1% NaN3 (Sigma) and resuspended in ice-cold HBSS/BSA/NaN3 at 5 x 106 cells/ml. The secondary, FITC-conjugated goat anti-mouse antiserum was added, and the cells left for a 30-min incubation on ice in the dark. Following staining with the secondary, cells were washed with ice-cold HBSS/BSA/NaN3 and fixed in 1% paraformaldehyde. Fluorescent cells were counted using a Becton Dickinson FACS Calibur flow cytometer and analyzed with WinList 5.0 software (Verity Software House, Topsham, ME, USA).

Statistical analysis
Data were analyzed using one-way ANOVA followed by Tukey’s post hoc test. Statistical analyses were conducted using SPSS Version 10 (SSPS Inc., Corona, CA, USA).


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RESULTS
 
PLD inhibition by butanol suppresses up-regulation of the Mac-1 activation epitope
PLD activation is linked with a number of granulocyte activation events, including the oxidative burst, degranulation, and expression of the Mac-1 activation epitope in eosinophils [23 ] and with fMLP stimulation of neutrophils [18 ]. Primary alcohols remain as the choice of PLD inhibitor in primary granulocytes; in the presence of primary alcohols, PLD catalyzes a transphosphatidylation reaction resulting in the generation of inactive phosphatidylalcohol, instead of phosphatidic acid (Fig. 1 ). Thus, we used 1-butanol to indicate whether PLD activation is involved in the up-regulation of the Mac-1 activation epitope by C5a and fMLP. Figure 2A shows that some neutrophils in the enriched but otherwise untreated population express the activation epitope, detected using CBRM1/5, compared with staining using an isotype control. The gate, R3, is set with 2% of the untreated isotype control stained cells considered positive. The number of positive neutrophils increases with exposure to C5a or fMLP, albeit to a greater extent using fMLP. Incubating the neutrophils in 1-butanol dose-dependently prevented the staining, with a concentration of 0.5% reaching a maximal effect. This is the same dose chosen by others to be optimal for impeding neutrophil migration to fMLP in a flow chamber paradigm [18 ]. Neither tert-butanol nor 1-butanol used at 0.5% affected neutrophil viability measured by Trypan blue exclusion (data not shown). Figure 2B shows the inhibition of expression averaged from six different neutrophil donors and includes data for the control alcohol, tert-butanol, also used at 0.5%. C5a and fMLP also cause an up-regulation of the total level of surface expressed Mac-1 and 0.5% 1-butanol prevented this increase (data not shown). Expression of the Mac-1 activation epitope is correlated with the capacity of neutrophils to adhere to a Mac-1 ligand [29 ] and knockdown studies in granulocyte differentiated HL60 cells implicated PLD in phagocyte adhesion [25 ]. We determined that TNF-stimulated neutrophil adhesion to the Mac-1 ligand, fibrinogen, is inhibited with 1-butanol (data not shown). Taken together, these results indicate that PLD is active and required for expression of the Mac-1 activation epitope stimulated by C5a, similar to expression stimulated by fMLP.


Figure 1
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  		Figure 1. Primary alcohols block generation of phosphatidic acid by substituting as a substrate for PLD.


Figure 2
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  		Figure 2. PLD is required for the expression of the Mac-1 activation epitope. (A) Flow cytometry using monoclonal antibody CBRM1/5 was employed to detect the Mac-1 activation epitope on neutrophils [29 ]. Enriched but otherwise untreated neutrophils (labeled "no treatment") show roughly that 15% of the cells bear the activation epitope compared with the isotype control antibody arbitrarily set to include only 2% of cells in the gated region, R3. The number of neutrophils expressing the epitope, e.g., moving into R3, is increased by both C5a and fMLP, and in each case, this expression is dose-dependently inhibited by incubation in 1-butanol. (B) Pooled flow cytometry data from 6 donors were converted to percent inhibition and calculated as follows: 1 (number of positive neutrophils with chemoattractant in 0.5% alcohol/number without alcohol) x 100%. The inhibition is significantly greater with 1-butanol. The mean and standard error is shown.

Inhibition of the PLD pathway does not affect neutrophil chemotaxis across acellular filters
We next investigated whether neutrophil chemotaxis across acellular Transwell filters is affected by incubation in the alcohols, or alternatively, whether chemotaxis is dependent on PLD. Shown in Fig. 3A , neither the control tert-butanol nor the 1-butanol used at 0.5% significantly affected neutrophil chemotaxis across acellular filters. To distinguish the primary alcohol effects from nonspecific effects of the control alcohol, we calculated the chemotaxis in the presence of 1-butanol in relation to chemotaxis in the presence of tert-butanol. Figure 3B shows that even using this ratio, the highest dose of 1-butanol did not reach statistical significance in terms of preventing migration. We interpreted this to mean that migration across acellular filters is PLD and Mac-1-independent, which is compatible with other studies, including results using neutrophils of patients with Leukocyte Adhesion Deficiency, which still exhibited chemotaxis [31 ].


Figure 3
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  		Figure 3. Role of PLD in neutrophil chemotaxis across bare filters. (A) Effect of butanols on neutrophil chemotaxis in response to C5a and fMLP across acellular filters. Neutrophils were left untreated or incubated with 0.5% 1-butanol or tert-butanol for 20 min at room temperature, then added, unwashed, to the top chamber of Transwell filters and induced to migrate with 10–9 M C5a or 10–8 M fMLP for 70 min. Bars represent the mean percentage of cells recovered in the lower chamber compared with the number of cells added to the top chamber at the beginning of the assay. Each bar is the mean of the mean from 3 experiments ± SEM, with triplicate wells used in the mean for each experiment. (B) Lack of effect of 1-butanol on neutrophil migration across acellular filters. Neutrophils were incubated with different concentrations of 1-butanol or tert-butanol for 20 min at room temperature and then added to the top chamber of Transwell filters and induced to migrate across acellular filters as described above. Bars represent the percentage of cells migrating in the presence of 1-butanol relative to the same concentration of tert-butanol. Each bar is the mean of the mean from 4 experiments ± SEM.

Neutrophils but not T84 cells express PLD1
Human neutrophils constitutively express mRNA variants of PLD1 and PLD2 with PLD1 in greater abundance. PLD1 protein is also found in greater abundance than PLD2 protein [32 ]. Thus we focused our effort on detecting PLD1 and using immunoprecipitation followed by Western blot analysis, we detected one principal band and multiple fainter bands of similar molecular weight in neutrophil lysates (Fig. 4 ). This is contrasted with the T84 epithelial cell line in which no similarly sized bands were detected, suggesting that PLD1 is not expressed by this line. We therefore proceeded to test the impact of 1-butanol on neutrophil transepithelial migration, presuming that any effect is unlikely to be due to inhibition of PLD in the epithelial cells.


Figure 4
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  		Figure 4. Neutrophils but not T84 cells express PLD1. Cell lysates were directly prepared for SDS-PAGE (total lysate) or incubated with antibody to PLD1, and the precipitated product was separated by SDS-PAGE, blotted to nitrocellulose, then probed using the same antibody (immuneppt.). A band corresponding with the predicted size of PLD is evident in the neutrophil lane of three donors (S1–S3) but not the T84 lane.

1-butanol inhibits neutrophil migration across T84 monolayers in response to fMLP but not C5a
Blocking PLD activity did not significantly affect neutrophil chemotaxis across acellular filters, but this does not rule out a possible impact on neutrophil interactions unique with epithelial cells, especially since migration through an epithelial barrier to fMLP is Mac-1 dependent. Figure 5A shows that neither alcohol had a significant blocking effect on neutrophil migration across T84 monolayers to C5a but that the tert-butanol (0.5%) inhibited as much as 40% of neutrophil transepithelial migration in response to fMLP while 1-butanol inhibited up to 80% of migration. When migration was represented as a ratio of the two alcohols a significant amount of neutrophil transepithelial migration to fMLP could be attributed to the specific inhibition (of PLD) by 1-butanol (Fig. 5B) . Inhibition of migration in response to fMLP did not result in increased numbers of adherent neutrophils (data not shown). Although the lack of inhibition by the alcohols on C5a-mediated migration suggested there was no effect of the butanols on the epithelial cells, we tested the possibility that the butanols influenced transcellular diffusion by measuring monolayer permeability to phenol red (spiked into dye-free media and read spectroscopically). Approximately 18% of the dye placed in the top chamber with the neutrophils diffused into the bottom chamber by the end of the assay; this amount of diffusion was similar in all treatment groups (data not shown). Finally, we examined whether the effect of 1-butanol was transient, possibly being relieved as the alcohols diffused into the lower chamber; however, the inhibitory effect of 1-butanol on neutrophil migration persisted for up to 3 h, 1 h longer than our typical assay time and the longest time period we examined. This pattern of results suggests that PLD activation is critical during fMLP- but not C5a-mediated neutrophil-epithelial interactions.


Figure 5
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  		Figure 5. Role of PLD in neutrophil migration across inverted T84 monolayers. (A) Effect of tert- and 1-butanol on neutrophil migration across T84 intestinal epithelial monolayers. Neutrophils were left untreated or incubated with 0.5% tert-butanol or 0.5% 1-butanol as described in Fig. 2 and induced to migrate across inverted T84 monolayers with 10–8 M C5a or 10–7 M fMLP for 2 h. Bars represent the percentage of cells added to the top chamber at the beginning of the migration assay. Each bar is the mean from 4 experiments ± SEM for C5a, and 9 experiments ± SEM for fMLP. (B) Dose-dependent effect of 1-butanol on neutrophil migration across T84 monolayers. Similar to A, neutrophils in a range of alcohol concentrations were induced to migrate to C5a or fMLP over a period of 2 h. Bars represent the mean of the mean percentage of migrated cells in the presence of 1-butanol relative to the same concentration of tert-butanol from 4 experiments ± SEM. (C) Migration time course in the presence of 0.5% butanol treatment. Neutrophil migration across inverted T84 monolayers in the presence of 0.5% 1-butanol or tert-butanol was performed as above. The solid line is migration in response to C5a; the broken line is migration in response to fMLP. Each time point is the mean percentage of migrating cells treated with 1-butanol relative to the tert-butanol ± SD of three replicates from a single donor.

PA increases neutrophil migration to fMLP blocked by 1-butanol
The breakdown by PLD of phosphatidylcholine results in the generation of PA (Fig. 1) . We therefore wanted to determine whether applying exogenous PA would restore neutrophil transepithelial migration to fMLP blocked by 1-butanol. To optimize exogenous PA reaching the cytoplasm, we used the medium-chain water-soluble DiC8-PA. The addition of PA did not directly result in increased surface expression of total Mac-1 (data not shown), and exogenous PA was unable to directly initiate neutrophil transepithelial migration, e.g., in the absence of a chemoattractant (Fig. 6A , black bars); however, it did result in increased neutrophil adhesion to the T84 monolayers in a dose-dependent manner (Fig. 6A , open bars). On the other hand, in the presence of a fMLP gradient, DiC8-PA significantly increased migration of neutrophils preincubated with 1-butanol (Fig. 6B) . The experiments including fMLP were conducted using 50 µM DiC8-PA; higher concentrations were inhibitory to migration due to increased adhesion. Neutrophil migration in response to C5a was unaffected by DiC8-PA unless concentrations higher than 50 µM were used (data not shown).


Figure 6
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  		Figure 6. Phosphatidic acid (PA) increases neutrophil adhesion to inverted T84 monolayers and migration to fMLP blocked by 1-butanol. (A) Effect of PA on neutrophil chemokinesis across inverted T84 monolayers lacking chemoattractants. Neutrophils were incubated in the respective concentration of DiC8-PA then applied to the top chambers of inverted T84 monolayers and incubated for 2 h. Cells from the bottom chamber (migrated, black bars) as well as monolayer-associated cells (adherent, open bars) were collected. Each bar is the mean of migration from 3 wells ± SD of a single donor. The experiment was repeated with two other donors with a similar pattern in the results. (B) Migration of neutrophils treated with 50 µM DiC8-PA across the inverted T84 monolayers in response to fMLP. Each bar is the mean of the mean percentage migrated cells from 4 experiments ± SEM.

PA does not increase migration of Mac-1-deficient dbcAMP-differentiated HL-60 cells
We have previously shown that promyelocytic HL-60 cells differentiated with dbcAMP into neutrophil-like cells (dHL-60) and despite expressing CD18, lack Mac-1 expression and do not effectively migrate across epithelial monolayers in response to fMLP [9 ]. To further confirm that the mechanism of PA-restored neutrophil migration in response to fMLP is attributable to restored Mac-1 function, we treated dHL-60 with DiC8-PA. As predicted, and shown in Fig. 7 , exogenous DiC8-PA did not increase migration of the Mac-1-deficient cells in response to fMLP.


Figure 7
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  		Figure 7. Migration of dbcAMP-differentiated HL-60 cells across inverted T84 monolayers in the presence of added phosphatidic acid. HL-60 cells were differentiated into the neutrophil-like cells as described in Materials and Methods and characterized elsewhere [9 ]. Cells were left untreated (black bars), or treated with 50 µM (gray bars) or 100 µM (hollow bars) DiC8-PA for 10 min at room temperature. Migration across T84 monolayers was induced by 10–8 M C5a or 10–8 M fMLP. Each bar is the mean of the mean percent of applied cells from 3 experiments ± SEM.

PA but not diacylglycerol is involved in neutrophil transepithelial migration in response to fMLP
PA can signal by interacting with a number of other molecules or by stimulating tyrosine phosphorylation. Additionally, PA can be further converted into diacylglycerol (DAG) by PA phosphohydrolase (Fig. 1) . Some investigators, therefore, attribute the effects of PLD activation to DAG, while others claim that PA can mediate proinflammatory functions independent of the generation of DAG. To distinguish whether neutrophil transepithelial migration in response to fMLP is dependent on DAG, we treated neutrophils with propranolol, an inhibitor of PA phosphohydrolase. Propranolol at concentrations up to 300 µM did not significantly inhibit neutrophil transepithelial migration (Fig. 8 ), suggesting that PA but not PA-derived DAG is important in neutrophil transepithelial migration in response to fMLP.


Figure 8
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  		Figure 8. Phosphatidic acid phosphohydrolase inhibitor propranolol does not inhibit neutrophil migration across inverted T84 monolayers. Neutrophils were incubated with 300 µM propranolol for 20 min, and the migration assay was performed as described above. Bars represent the mean of the mean percentage of applied cells from 4 (for C5a) or 5 (for fMLP) independent experiments ± SEM. The difference between no treatment and propranolol treatment groups did not reach statistical significance for either C5a or fMLP.

Other PLD-dependent functions are not involved in neutrophil transepithelial migration in response to fMLP and C5a
Our results show that neutrophil transepithelial migration in response to C5a is independent of Mac-1 and PLD and therefore independent of other downstream PLD-dependent functions; however, this has not been confirmed to be the case when fMLP is the chemoattractant. NADPH oxidase activation and degranulation are reportedly dependent on PLD [33 ] and therefore may be important in fMLP-directed transmigration. We therefore wanted to determine whether neutrophil transintestinal epithelial migration in response to fMLP is dependent on the oxidative burst. Treating neutrophils with superoxide dismutase and catalase did not inhibit migration in response to fMLP or C5a, and second, neutrophils from a chronic granulomatous disease patient migrated in response to fMLP and C5a (data not shown), suggesting that superoxide production is not required. Finally, exogenous PA facilitated transmigration without increasing total surface Mac-1 indirectly indicating that degranulation does not play a role in neutrophil transepithelial migration. Taken together, these data allowed us to conclude that neither degranulation nor the oxidative burst is involved in neutrophil transintestinal epithelial migration to either chemoattractant. We conclude that regulating Mac-1 binding through the activation epitope is the primary mechanism influenced by PLD during neutrophil migration across intestinal epithelium in response to fMLP and that this is not critical to cell migration to C5a.


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DISCUSSION
 
Neutrophils have been shown to migrate into the intestinal lumen during active inflammation and appear as crypt abscesses [34 , 35 ]. Although the chemotactic signal responsible is unknown, the great abundance of bacteria in the lumen would favor the idea that formylated peptides recruit neutrophils into the lumen. However, small formylated peptides are also present in the colon lumen of healthy individuals, yet there is no chronic inflammatory infiltrate. In fact, a colonocyte line was recently reported to possess formylated peptide receptors [36 ], a finding that will require a reevaluation of the impact of these peptides in epithelial pathobiology. Therefore some consideration of alternate candidate chemoattractants is needed. From human studies, IL-8 and LTB4 have been detected in the lumen during intestinal inflammation; however, these may be derived from neutrophils already present and may not represent early-stage chemoattractants. On the basis of multiple lines of evidence, we are suggesting that activated complement contributes to the early recruitment of neutrophils into the lumen. Intestinal epithelial cells have been shown to synthesize complement components [37 , 38 ] and complement activation and deposition of IgG have been detected on the luminal side of the epithelium in the colon of patients with ulcerative colitis [39 ], showing that complement cleavage occurs in the lumen. Decay accelerating factor (DAF), a protein that controls the C5 convertase, is expressed on the lumenal side of intestinal epithelial cells, inferring protection from complement activation through to the membrane attack complex in the lumen is important [40 , 41 ]. Complementary to observations from humans are the findings that mice inflamed with dextran sodium sulfate show activated complement products in their colonic mucosa and that DAF gene knockout mice experience an exacerbated colitis with greater immune complex deposition [42 ]. Finally, antagonizing C5a reportedly attenuates experimental colitis, though the critical site of activation was not determined [43 ]. Thus there are other possible candidate chemoattractants, including complement products, to recruit neutrophils into the lumen, and we have begun to compare mechanisms of neutrophil transepithelial migration to these chemoattractants with fMLP.

In the course of exploring transepithelial migration to various chemoattractants, we discovered that neutrophils can use Mac-1–independent mechanism(s) in response to C5a, IL-8, and LTB4 [8 ]. That Mac-1-independent neutrophil recruitment occurs in vivo was reported using CD11b-deficient mice with DSS colitis, which experienced exacerbated disease compared with wild-type mice [10 ]. These discoveries have impelled us to better define the mechanisms of Mac-1-independent transepithelial migration, including which adhesion molecules are involved and whether they employ second-messenger signaling mechanisms different from the fMLP receptor. Now, we determined that indeed, neutrophil migration in response to the chemoattractant C5a is PLD-independent, whereas migration in response to fMLP is PLD-dependent, due to the regulation of the Mac-1 activation state by PLD.

We found that neutrophil chemotaxis in response to both C5a and fMLP across acellular filters was not significantly inhibited by 1-butanol and are therefore PLD-independent (Fig. 3) . Similarly, neutrophil and neutrophil-differentiated HL-60 cell transintestinal epithelial migration in response to C5a was also PLD-independent; however, blocking the PLD pathway with 0.5% 1-butanol specifically inhibited up to 75% of neutrophil migration in response to fMLP (Fig. 5) . This pattern is compatible with neutrophil transepithelial migration in response to fMLP being dependent on Mac-1, including early adhesive interactions [21 , 44 , 45 ]. As there was no significant increase in adherent neutrophils when PLD was blocked, a PLD-dependent interaction is likely among the earliest steps in neutrophil interactions with epithelial cells. Moreover, that the Mac-1 dependency of transepithelial migration in response to fMLP is due to PLD activation is supported by our results directly showing that the activation epitope is regulated by PLD. We found that both neutrophil expression of the Mac-1 activation epitope (Fig. 2) and adhesion to fibrinogen are inhibited by 1-butanol. Finally, the addition of DiC8-PA resulted in a dose-dependent increase in neutrophil adhesion to fibrinogen (not shown) and significantly restored neutrophil migration in response to fMLP inhibited by 1-butanol (Fig. 6B) . Yet exogenous PA did not restore increased Mac-1 surface expression elicited by fMLP, which supports the idea that affinity not the total Mac-1 is important in adhesion and transepithelial migration. Together, our data suggest that modulating Mac-1 activation is the likely outcome of PLD activation during neutrophil transepithelial migration in response to fMLP. On the other hand, neutrophil migration in response to C5a, being primarily Mac-1-independent, is not blocked by 1-butanol and therefore is independent of PLD activation of the Mac-1 epitope.

Neutrophil migration across T84 monolayers in response to infection with S. typhimurium is reportedly PLD and DAG dependent. PLD activation results in the apical release of the chemoattractant Hepoxilin A3 by infected epithelial cells, yet Hepoxilin A3 is not involved in neutrophil transepithelial migration in response to fMLP [46 , 47 ]. These findings then are compatible with migration to Hepoxilin A3 likely being Mac-1 dependent, similar to fMLP, except that propranolol did not significantly inhibit fMLP-mediated migration in our experiments (Fig. 8) . Thus migration to fMLP depends on PA signaling events but not DAG.

Showing that migration to C5a is strongly PLD independent, albeit indirectly, suggests that neutrophil transepithelial migration to this chemoattractant is also independent of other effector activities of PLD, such as degranulation and oxygen radical generation. This seems to be the case for fMLP-mediated migration as well; it is independent of events downstream of PLD activation. First, incubation in propranolol, intended to inhibit DAG, had no effect on migration, ruling out downstream effector events. Additionally, others have reported, and we have repeated, that cells from a chronic granulomatous patient migrate efficiently across T84 monolayers to fMLP and C5a (unpublished data), which rules out a role for oxygen radicals [48 ].

The use of butanols to study PLD may be criticized as nonspecific yet remains the inhibitor of choice for use with primary leukocytes. Alcohols present during the migration assay might also be affecting epithelial PLD function, or nonspecifically acting on the epithelial cells to impede migration. There is a report that endothelial PLD is activated during neutrophil transmigration, and PLD activation increases the permeability of endothelial monolayers [49 ]. We did not detect PLD1 in the T84 cells (Fig. 4) , nor did we find changes in monolayer permeability measured by 125I-HSA or phenol red diffusion in the presence of either alcohol (data not shown). Finally, neutrophil migration in response to C5a was unaffected by PLD blockade using butanol, suggesting that any epithelial PLD is unlikely to be involved in neutrophil transmigration.

Considering that no single chemoattractant has been shown responsible for crypt abscesses, it is possible that different chemoattractants are recruiting neutrophils in different IBD patients, perhaps related to specific disease etiologies or stages of inflammation. It is also possible that different combinations of chemoattractants are present in various disease stages in the same patient. For example, increased intestinal permeability in some patients might account for the breach of epithelial integrity, and the initial translocation of bacteria or bacterial products leads to neutrophil recruitment; in turn, these neutrophils become a source of chemoattractant from within the lumen. Perhaps in patients without compromised barrier function, the deposition of antibody against an apical epithelial antigen and complement activation on the lumen side of epithelium may be part of the disease pathogenesis [39 ]. Cleavage of C5 to C5a, in turn, may play a role in neutrophil recruitment into the lumen, with the process of transmigration affecting the barrier. Our data cannot rule out that both Mac-1 and PLD-dependent and -independent mechanisms of neutrophil recruitment are critical in human colitis, although it is clear that colitis includes neutrophil infiltrates in CD11b gene knockout mice [10 ].

In summary, we show that neutrophil chemotaxis across acellular filters and transintestinal epithelial migration in response to C5a are PLD independent, while neutrophil transepithelial migration in response to fMLP but not C5a is PLD dependent, suggesting that PLD is important for the fMLP-activated neutrophil interaction with epithelium (summarized in Fig. 9 ). Regulation of Mac-1 expression, specifically, the Mac-1 affinity, but not other PLD-dependent events such as neutrophil superoxide production or degranulation or epithelial chemoattractant release, is involved in neutrophil migration in response to fMLP. Anti-inflammatory strategies must address the alternative pathways in order to effectively prevent neutrophil recruitment into the lumen.


Figure 9
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  		Figure 9. PLD, though activated by both C5a and fMLP, distinguishes Mac-1-dependent from Mac-1-independent neutrophil migration across intestinal epithelium.


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ACKNOWLEDGEMENTS
 
This study was supported by grants from the Crohn’s and Colitis Foundation of Canada and the Natural Sciences and Engineering Research Council of Canada (NSERC). S. Carrigan was supported by a scholarship from NSERC and an Isaac Walton Killam Health Centre Graduate Scholarship. We would like to thank Hana James for her technical expertise and Drs. A. Issekutz, J. Blay, D. Byers, and C. McMaster (all at Dalhousie University) for their insights and help.

Received August 22, 2006; revised July 27, 2007; accepted August 7, 2007.


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